Bacillus cereus is a gram-positive, rod-shaped, beta-hemolytic bacterium. It occurs widely in nature, being found in soil and in food, and is known to be a food-borne human pathogen causing emesis or diarrhea. The toxin produced by Bacillus cereus is known as Bacillus Diarrheal Enterotoxin (BDE), a three-component cytotoxin consisting of Hemolysin BL (Hbl), Nonhemolytic Enterotoxin (Nhe) and Cytotoxin K (CytK). Nhe is one of the major causes of non-hemolytic food poisoning.
The pancreas gland in mammals secretes a variety of enzymes and variety of substances, including proteases such as trypsinogen, chymotyrpsinogen, elastase and carboxypeptidase, peroxidase, pancreatic lipase, and amylase. Without these enzymes (normally produced by the human pancreas), a substantial portion of undigested food simply passes through the digestive tract and provides no nutritional benefit. The pancreatin/pancrelipase active pharmaceutical ingredient (API) is an enzymatically active product derived from porcine pancreas glands. Porcine pancreatin juice is closest to that of humans, with high proportions of lipase and alpha-amylase in comparison to other mammals. Therefore, pancreatin/pancrelipase is made from the pancreas of pigs, and is used to treat conditions in which pancreatic secretions are deficient, such as surgical pancreatectomy, pancreatitis and cystic fibrosis. It has been claimed that pancreatin is beneficial in the treatment of food allergies, celiac disease, autoimmune disease, cancer and weight loss. Pancreatin is sometimes called “pancreatic acid”, although it is neither a single chemical nor an acid.
Pancreatin enzyme products (PEPs) of porcine or bovine origin have been available in the United States for the treatment of exocrine pancreatic insufficiency (EPI) since before the enactment of the Federal Food, Drug, and Cosmetic Act of 1938 (the Act). With the exception of one PEP approved in 1996, the products have been marketed without New Drug Applications (NDAs) and were considered as dietary supplements. In both Europe and the US, the use of PEPs has been severely restricted in recent years, due to concerns about the products' origin from animal tissue (with concomitant risk of viral disease transmission) and the relatively poor characterization and lack of standardization of enzymatic bioavailability in humans. The Food and Drug Administration (FDA) determined that an Over The Counter (OTC) monograph would not be sufficient to adequately regulate these drug products or to standardize enzyme bioactivity, safety and effectiveness. The FDA's guidance for the industry requires all pharmaceutical companies marketing pancreatic enzymes for pancreatin deficiency to be approved under New Drug Applications. Since April 2010, PEPs are only available by prescription and only PEPs approved by the FDA remain on the market.
To be approved, an NDA must meet the requirements in 21 CFR §314.50 regarding chemistry, manufacturing and controls (CMC) information. The drug substance should be adequately characterized using chemical, physical and biological testing methods. Batch-to-batch consistency with respect to chemical identity, biological activity of different classes of enzymes including specific activity, and identity and purity levels should be demonstrated. Since Pancreatin is referenced in a new NDA, the agency expects the pancreatin Drug Master File (DMF) to meet current ICH Q6B requirements for specifications. Specifications for the drug substance should include tests for identity, biological activity of different classes of enzyme, purity and other relevant attributes. Because of the complexity of pancreatin extract product, it is unlikely that currently-available physiological and biological analytical tools would be able to demonstrate that the active ingredients in pancreatic extract products from two different batches/manufacturers are the same. Current United States Pharmacopeia (USP) monograph tests are insufficient to characterize the API to meet the ICH guideline. The new regulatory guidelines now require better methods to characterize pancreatin API.
3M's TECRA® Assay is an enzyme-linked immunosorbent assay (ELISA) normally used for determining the absence or presence the presence of bacterial toxins from organisms such as Bacillus cereus in food and food-related products. It can, with some difficulty, be modified and adapted to test more complex biologically-derived products such as pancreatin for the absence or presence of BDE. Even when so modified and adapted it remains prone to false positive and false negative results. When the TECRA® assay is used for the testing of complex protein-dense mixtures such as pancreatin, non-specific binding of its antibodies to porcine proteins is clearly evident and contributes to difficulties in its routine use.
Therefore, a more reliable assay is desired for determining the absence or presence of Bacillus cereus in a sample that minimizes or eliminates altogether the false positives and false negatives. The assay should be at least as sensitive as the TECRA® assay for the detection of BDE in a positive control sample.